Question

describe the overall organization (domains) of a DNA polymerase. on a diagram show its active site and describe the mechanism of the reaction. why does nucleotide polymerization have downhill free energy?

Answer 1

DNA polymerase couples chemical energy to translocation along a DNA template with a specific directionality while it replicates genetic information. According to single-molecule manipulation experiments, the polymerase-DNA complex can work against loads greater than 50 pN. It is not known, on the one hand, how chemical energy is transduced into mechanical motion, accounting for such large forces on sub-nanometer steps, and, on the other hand, how energy consumption in fidelity maintenance integrates in this non-equilibrium cycle. Here, we propose a translocation mechanism that points to the flexibility of the DNA, including its overstretching transition, as the principal responsible for the DNA polymerase ratcheting motion. By using thermodynamic analyses, we then find that an external load hardly affects the fidelity of the copying process and, consequently, that translocation and fidelity maintenance are loosely coupled processes. The proposed translocation mechanism is compatible with single-molecule experiments, structural data and stereochemical details of the DNA-protein complex that is formed during replication, and may be extended to RNA transcription.

A polymerase is a motor protein that transfers genetic information inside biological cells. There exist two types of polymerases, DNA and RNA polymerase (DNAp and RNAp)1, 2. The former replicates a DNA template strand into a complementary DNA strand, a process known as replication that is needed for cell division. The latter transfers the information of a DNA template into an RNA transcript in the so-called transcription process. The product of replication is a double-helix DNA molecule made up of two complementary strands and that of transcription is a double-helix hybrid molecule made up of a DNA strand and a complementary RNA strand. DNAp and RNAp translocate on a stepwise fashion on consecutive DNA template positions, hydrolyzing triphosphate into monophosphate nucleosides for their ultimate incorporation into the replicate or transcript strand. Although statistical and kinetic models have described the stochastic behavior and information processing by DNAp and RNAp3–8, it is not known how the chemical free energy obtained from this hydrolysis reaction is transduced into the polymerase mechanical motion.

In contrast, motion and force generation mechanism are clear in other motor proteins. Kinesin hydrolyzes ATP into ADP plus phosphate (Pi). ATP binding to the forward head attaches this head to the microtubule track and ATP hydrolysis in the rear head releases this head from the microtubule. After ATP hydrolysis, the protein can move either forward or backward by ratcheting and, on average, it performs a net forward step by bringing the rear head to the front by using the asymmetric torsional strain accumulated between its two tails in the helical stalk. Rotational steps of ATPases9, 10 or bacteriophage packaging mechanisms and force generation, see refs 11 and 12 for reviews, have also been analyzed. Kinesin withstands maximum forces of ≈7 pN under saturating ATP conditions13, a load limit that is related to the torsional strain that can be accumulated in the helical stalk, and moves in 8-nm steps, a length that is related, on the one hand, to the average separation between heads after a 180-degree rotation of one head over the other in its hand-over-hand movement and, on the other hand, to the tubulin-dimer length. which is a flexible structure. It moreover produces strain on this molecular track, unlike kinesin, whose microtubular track can be considered rigid with respect to the strength of the forces developed by this transport motor. The flexibility of double-stranded DNA (dsDNA) has been thoroughly characterized in single-molecule experiments Two almost linear elasticity regimes and a transition to an almost unwound state have been described for this polymer. At low forces (5 pN), the dsDNA aligns straight with the applied force; at higher forces, ≈5 − 65 pN, the polymer is stretched intrinsically. This regime of elasticity, which is thus known as intrinsic or enthalpic, can be characterized by a Young modulus, like a macroscopic material. At ≈65 pN the copolymer experiences an overstretching transition to 1.7 times its contour length.

DNA polymerase is an enzyme that synthesizes DNA molecules from deoxyribonucleotides, the building blocks of DNA. These enzymes are essential for DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones. These enzymes catalyze the chemical reactions

I

deoxynucleoside triphosphate + DNAn ⇌ diphosphate + DNAn+1.

DNA polymerase adds nucleotides to the three prime (3')-end of a DNA strand, one nucleotide at a time. Every time a cell divides, DNA polymerases are required to help duplicate the cell's DNA, so that a copy of the original DNA molecule can be passed to each daughter cell. In this way, genetic information is passed down from generation to generation.

Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form, in the process breaking the hydrogen bonds between the nucleotide bases. This opens up or "unzips" the double-stranded DNA to give two single strands of DNA that can be used as templates for replication.

Structure. The basic structure of all DNA polymerases consists of subdomains referred to as the palm, fingers, and thumb and resemble an open right hand. The palm contains catalytically essential amino acids in it's active sites. The fingers are essential for nucleotide recognition and binding.

DNA polymerase (pol) β is a small eukaryotic DNA polymerase composed of two domains. Each domain contributes an enzymatic activity (DNA synthesis and deoxyribose phosphate lyase) during the repair of simple base lesions. These domains are termed the polymerase and lyase domains, respectively. Pol β has been an excellent model enzyme for studying the nucleotidyl transferase reaction and substrate discrimination at a molecular level. In this review, recent crystallographic studies of pol β in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA (apurinic sites and 8-oxoguanine) are described. Structures of these catalytic intermediates provide unexpected insights into mechanisms by which DNA polymerases enhance genome stability. These structures also provide an improved framework that permits computational studies to facilitate the interpretation of detailed kinetic analyses of this model enzyme.

DNA polymerases catalyze template-dependent DNA synthesis during genome replication and repair. These enzymes are responsible for preferentially binding and incorporating a nucleotide, from a pool of chemically and structurally similar molecules, that correctly base pairs with the appropriate templating base. DNA polymerase (pol) β has served as a model enzyme for studying this fundamental task, providing a detailed understanding of the events during substrate selection. Pol β is the smallest cellular DNA polymerase (335 residues, 39 kDa) and lacks a 3′ → 5′ proofreading exonuclease activity that enhances the accuracies of replicative DNA polymerases (e.g., pol ε and pol δ). On the basis of its primary sequence, pol β belongs to the X-family of DNA polymerases.1 This review will highlight recent advances and insights provided by the structural characterization of pol β in various liganded and conformational states. For earlier kinetic and structural descriptions, see previous in-depth reviews.

Biological Role:Endogenous and environmental agents continually modify genomic DNA, resulting in physical damage or modification that results in steady-state levels of 50000—200000 apurinic/apyrimidinic (AP) sites per eukaryotic cell.4 AP sites are generated through spontaneous depurination or lesion specific enzymatic hydrolysis of the N-glycosyl bond between the deoxyribose and base. The rate of spontaneous depurination has been estimated to be ∼104 depurinations per cell per day.The base excision repair (BER) pathway (Figure ​(Figure1)1) is responsible for removing simple base lesions and AP sites in DNA. Pol β contributes two enzymatic activities, DNA synthesis and deoxyribose phosphate (dRP) lyase, during the repair of AP site

AP sites represent potentially dangerous lesions because they can be mutagenic and cytotoxic. AP endonuclease 1 incises the AP site, resulting in 3′-hydroxyl and 5′-dRP termini. The dRP group is excised by the lyase activity of pol β, resulting in a 5′-phosphate and a one-nucleotide gap (i.e., a single templating base). Pol β fills this single-nucleotide gap, resulting in nicked DNA that will subsequently be ligated to restore DNA’s native structure.

As discussed in detail below and elsewhere,pol β and other members of the X-family of DNA polymerases have evolved to fill short DNA gaps during essential cellular transactions. In a mouse model system, the loss of pol β results in embryonic lethality; however, cultured embryonic mouse fibroblasts are viable. These cells are hypersensitive to genomic toxicants because of the accumulation of cytotoxic repair intermediates. In addition to its enzymatic activities, pol β physically interacts with other key BER factors that hasten repair at AP sites.

The fundamental role that pol β plays in BER and high-fidelity gap-filling DNA synthesis implicates pol β and BER as tumor suppressors.Consistent with this idea is the observation that a high percentage of tumors have variants of pol β. These often have altered fidelity or catalytic activities and can induce cellular transformation.Many of these variants have amino acid changes that are distant from the polymerase or lyase active sites. It remains to be seen whether these alterations affect critical protein–protein interactions necessary for efficient BER or critical protein dynamic behavior that influences catalytic activity and/or fidelity

Polymerization of Nucleotides (Phosphodiester Bonds)

Nucleotides are joined together similarly to other biological molecules, by a condensation reaction that releases a small, stable molecule. Unlike proteins, carbohydrates, and lipids, however, the molecule that is released is not water but pyrophosphate (two phosphate groups bound together). When pyrophosphate is cleaved by the addition of water, a great deal of free energy is released, ensuring that the reverse process (hydrolysis of the phosphodiester bond to give free nucleotides) is very unlikely to occur.

hydroxyl group at the 2' position can participate in a reaction that cleaves the phosphodiester bond. Since this hydroxyl group is absent in DNA, the polymer is much more stable and lasts for a much longer time than it would with the hydroxyl. Thus, DNA can act as a stable long-term repository for genetic information. How stable? RNA is usually degraded within your cells in 30 minutes. On the other hand, DNA lasts your whole lifetime, and intact DNA thousands or millions of years old may be able to be recovered from frozen mammoth carcasses and mosquitoes trapped in amber.

Polynucleotides have a free 5' phosphate group at one end and a free 3' hydroxyl group at the other end. By convention, these sequences are named from 5' to 3'. For example, the molecule shown at right is ATC, not CTA.